Severe Peripheral Joint Laxity Is a Distinctive Clinical Feature of Spondylodysplastic-Ehlers-Danlos Syndrome (EDS)-B4GALT7 and Spondylodysplastic-EDS-B3GALT6

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Article Severe Peripheral Joint Laxity is a Distinctive Clinical Feature of Spondylodysplastic-Ehlers-Danlos Syndrome (EDS)-B4GALT7 and Spondylodysplastic-EDS-B3GALT6

Stefano Giuseppe Caraffi 1, Ilenia Maini 1,2, Ivan Ivanovski 1,3 , Marzia Pollazzon 1, Sara Giangiobbe 1, Maurizia Valli 4, Antonio Rossi 4, Silvia Sassi 5, Silvia Faccioli 5, Maja Di Rocco 6, Cinzia Magnani 7, Belinda Campos-Xavier 8, Sheila Unger 8, Andrea Superti-Furga 8,* and Livia Garavelli 1,* 1 Medical Genetics Unit, Maternal and Child Health Department, Azienda USL-IRCCS of Reggio Emilia, 42122 Reggio Emilia, Italy; stefanogiuseppe.caraffi@ausl.re.it (S.G.C.); [email protected] (I.M.); [email protected] (I.I.); [email protected] (M.P.); [email protected] (S.G.) 2 Child Neuropsychiatry Unit, Azienda USL of Parma, 43125 Parma, Italy 3 Department of Surgical, Medical, Dental and Morphological Sciences with interest in Transplant, Oncology and Regenerative Medicine, University of Modena and Reggio Emilia, 42121 Reggio Emilia, Italy 4 Department of Molecular Medicine, Unit of Biochemistry, University of Pavia, 27100 Pavia, Italy; zuff[email protected] (M.V.); [email protected] (A.R.) 5 Rehabilitation Pediatric Unit, Azienda USL-IRCCS of Reggio Emilia, Reggio Emilia, 42122 Reggio Emilia, Italy; [email protected] (S.S.); [email protected] (S.F.) 6 Department of Pediatrics, Unit of Rare Diseases, IRCCS Istituto Giannina Gaslini, 16147 Genoa, Italy; [email protected] 7 Neonatology and Neonatal Intensive Care Unit, Maternal and Child Department, University of Parma, 43121 Parma, Italy; [email protected] 8 Division of Genetic Medicine, Centre Hospitalier Universitaire Vaudois (CHUV), University of Lausanne, 1011 Lausanne, Switzerland; [email protected] (B.C.-X.); [email protected] (S.U.) * Correspondence: [email protected] (A.S.-F.); [email protected] (L.G.); Tel.: +41-(0)21-314-92-38 or +41-(0)21-314-00-14 (A.S.-F.); +39-0522-296244 (ext. 295463) (L.G.); Fax: +41-(0)21-314-35-46 (A.S.-F.); +39-0522-296266 (L.G.) Received: 13 August 2019; Accepted: 10 October 2019; Published: 12 October 2019

Abstract: Variations in genes encoding for the enzymes responsible for synthesizing the linker region of proteoglycans may result in recessive conditions known as “linkeropathies”. The two phenotypes related to mutations in genes B4GALT7 and B3GALT6 (encoding for galactosyltransferase I and II respectively) are similar, characterized by short stature, hypotonia, joint hypermobility, skeletal features and a suggestive face with prominent forehead, thin soft tissue and prominent eyes. The most outstanding feature of these disorders is the combination of severe connective tissue involvement, often manifesting in newborns and infants, and skeletal dysplasia that becomes apparent during childhood. Here, we intend to more accurately deﬁne some of the clinical features of B4GALT7 and B3GALT6-related conditions and underline the extreme hypermobility of distal joints and the soft, doughy skin on the hands and feet as features that may be useful as the ﬁrst clues for a correct diagnosis.

Keywords: beta-1,3-galactosyltransferase 6 (B3GALT6); beta-1,4-galactosyltransferase 7 (B4GALT7); spondyloepimetaphyseal dysplasia with joint laxity (SEMDJL1; SEMDJL-Beighton type); Ehlers–Danlos syndrome (EDS); spondylodysplastic Ehlers–Danlos syndrome (spEDS); spEDS-B3GALT6; spEDS-B4GALT7; extreme laxity of distal joints; soft; doughy skin on the hands and feet

Genes 2019, 10, 799; doi:10.3390/genes10100799 www.mdpi.com/journal/genes Genes 2019, 10, 799 2 of 23

Genes 2019, 10, 799 2 of 21

1. Introduction 1. Introduction The extracellular matrix (ECM) of connective tissues like cartilage, bone, tendon, ligaments and skin Theis a extracellularcomplex interacting matrix (ECM)arrangement of connective of proteins, tissues glycoproteins like cartilage, and bone, proteoglycans tendon, ligaments (PGs) andthat skinforms is the a complex scaffold interacting of the human arrangement body and of also proteins, constitutes glycoproteins the vital andenvironment proteoglycans supporting (PGs) thatcell formsgrowth the and sca differentiation.ffold of the human PGs bodyare macromolecul and also constituteses that theare vitalcomplex environment in their supportingstructures and cell growthrepresent and an di importantfferentiation. component PGs are macromolecules of both the ECM that and are of complex cell membranes. in their structures PGs have and mechanical represent anand important osmotic componentfunctions but of both are thestrongly ECM andinvolved of cell membranes.in signaling PGspathways have mechanical controlling and cell-to-cell osmotic functionsinteractions but as are well strongly as cell involvedgrowth and in signaling differentiation pathways and controllingtissue repair cell-to-cell [1–3]. PGs interactions are composed as well of a ascore cell protein growth and and one diff erentiationor more glycosaminoglyca and tissue repairn [ 1(GAG)–3]. PGs chains are composed that are long of a corepolysaccharides protein and oneconsisting or more of glycosaminoglycanrepeating disaccharide (GAG) units. chains On the that ba aresis longof the polysaccharides composition of these consisting units, of the repeating PGs are disaccharidesubdivided into units. heparan On the sulfate basis of (HS) the composition PGs and ch ofondroitin these units, sulfate the PGs(CS)/dermatan are subdivided sulfate into (DS) heparan PGs sulfate[4]. The (HS) biosynthesis PGs and chondroitinof the GAG side sulfate chain (CS) begins/dermatan with sulfatethe creation (DS) PGsof a [common4]. The biosynthesis tetrasaccharide, of the a GAGso called side “linker”, chain begins covalently with the linked creation to the of ahydroxyl common group tetrasaccharide, of serine residues a so called on the “linker”, core protein covalently (O- linkedglycosylation). to the hydroxyl group of serine residues on the core protein (O-glycosylation). TheThe linkerlinker regionregion isis synthesizedsynthesized throughthrough aa coordinatedcoordinated mechanismmechanism involvinginvolving specificspecific glycosyltransferases.glycosyltransferases. ItIt starts starts with with the the transfer transfer of a xyloseof a xylose residue residue by xylosyltransferase by xylosyltransferase I or II (encoded I or II by(encodedXYLT1 ,by OMIM XYLT1 *608124,, OMIM and *608124,XYLT2 and, OMIM XYLT2 *608125),, OMIM followed*608125), byfollowed the addition by the of addition two galactose of two residuesgalactose by residues galactosyltransferase by galactosyltransferase I, encoded by B4GALT7I, encoded(OMIM by 604327)B4GALT7 and galactosyltransferase(OMIM 604327) and II encodedgalactosyltransferase by B3GALT6 II(OMIM encoded *615291) by B3GALT6 (Figure (OMIM1). Subsequently *615291) (Figure there will 1). beSubsequently a transfer of there glucuronic will be acida transfer by glucuronosyltransferase of glucuronic acid by Iglucuronosyltransferase encoded by B3GAT3 (OMIM I encoded *606374). by B3GAT3 The so-called (OMIM “linkeropathies” *606374). The areso-called recessive “linkeropathies” conditions caused are recessive by variations conditions in genes caused encoding by variations the enzymes in genes responsible encoding for thethe synthesisenzymes responsible of the linker for region the synthesis of PGs. of the linker region of PGs.

FigureFigure 1.1. A.. GalactosyltransferasesGalactosyltransferases I (B4GALT7) andand IIII (B3GALT6)(B3GALT6) catalyzecatalyze thethe additionaddition ofof galactosegalactose unitsunits atat specificspecific positionspositions of of the the tetrasaccharide tetrasaccharide region region linking linking Serine Serine residues residues on on the the protein protein backbone backbone to B glycosaminoglycanto glycosaminoglycan (GAG) (GAG) chains. chains. Xyl = XylXylose, = Xylose Gal =,Galactose, Gal = Galactose, GlcA = Glucuronic GlcA = Glucuronic Acid. . Functional Acid. B. defects of B4GALT6 or B4GALT7 inhibit addition of one or both Gal monosaccharides, respectively, Functional defects of B4GALT6 or B4GALT7 inhibit addition of one or both Gal monosaccharides, and prevent GAG chain polymerization. respectively, and prevent GAG chain polymerization. Variants of the XYLT1 gene are responsible for skeletal dysplasias, including Baratela–Scott Variants of the XYLT1 gene are responsible for skeletal dysplasias, including Baratela–Scott syndrome (OMIM 615777, formerly erroneously called Desbuquois dysplasia type 2) [5–10], variants syndrome (OMIM 615777, formerly erroneously called Desbuquois dysplasia type 2) [5–10], variants of XYLT2 are the cause of the spondylo-ocular syndrome (OMIM 605822) [11–13], while variants of of XYLT2 are the cause of the spondylo-ocular syndrome (OMIM 605822) [11–13], while variants of B3GAT3 cause a Larsen-like syndrome and a more severe skeletal dysplasia (OMIM 245600) [14–20]. B3GAT3 cause a Larsen-like syndrome and a more severe skeletal dysplasia (OMIM 245600) [14–20]. In particular, variants in the B4GALT7 and B3GALT6 genes are the cause of a combined skeletal In particular, variants in the B4GALT7 and B3GALT6 genes are the cause of a combined skeletal and and connective tissue phenotype. connective tissue phenotype. A B4GALT7-related condition was initially described as “progeroid type 1” Ehlers–Danlos A B4GALT7-related condition was initially described as “progeroid type 1” Ehlers–Danlos syndrome (EDS), due to the phenotype characterized by premature aging and loose elastic skin in syndrome (EDS), due to the phenotype characterized by premature aging and loose elastic skin in one reported patient [21–30]. Subsequently, Cartault et al. in 2015 described a unique homozygous B4GALT7 mutation, p.Arg270Cys, in a large cohort of patients affected by the so-called Larsen Genes 2019, 10, 799 3 of 21 of Reunion Island syndrome [31]. This contributed to define the currently known phenotype of B4GALT7-related EDS, with round flat face, proptosis, short stature, hypotonia, radioulnar synostosis, osteopenia, hyperextensible skin and joint hypermobility (OMIM #130070) [24,25,31–33]. B3GALT6 variants cause a single disorder that was alternatively described as spondyloepimetaphyseal dysplasia (SEMD) with joint hypermobility (SEMDJL1 or SEMDJL Beighton type; OMIM #271640) in the “Nosology and classification of genetic skeletal disorders: 2015 revision” [34–43] or as severe EDS-like disorder (OMIM #615349) because of the striking joint laxity and muscular hypotonia in infancy [44,45]. Affected patients generally present clinical features of connective tissue weakness in infancy, and subsequently develop signs of skeletal dysplasia; sometimes these signs are already present at birth. Some patients develop life-threatening complications, such as aortic dilatation, aneurysms and cervical spine instability. Due to the mixed phenotype, with signs of EDS and signs of skeletal dysplasia, the B4GALT7 and B3GALT6-related syndromes have been called ”spondylodysplastic EDS” (spEDS) in the recently revised EDS classification of 2017 [46–48]. Hereby we present the clinical features of 3 patients with spEDS. Two are novel patients: Pt. 1 with B4GALT7-related EDS, and Pt. 3 with a severe B3GALT6-related EDS. Pt. 2 was originally reported as B3GALT6-related SEMDJL by Nakajima et al. in 2013, but is included here because his clinical description was largely unpublished and his condition has undergone a significant evolution. Through the description of these patients and a review of previous literature reports, we contribute to a more accurate definition of the clinical features associated with B4GALT7- and B3GALT6-related syndromes. In particular, we point out the extreme distal joint hypermobility and skin hyperextensibility (more evident in hands and feet) these two conditions have in common, and the peculiar radiological signs of each syndrome. The combination of these features is the main indicator for clinicians to suspect these ultra-rare conditions.

2. Materials and Methods

2.1. Metabolic Labeling of Fibroblast Cultures and PG Synthesis Analysis Skin fibroblasts from the patient and controls were cultured in minimum essential medium (MEM) with 10% fetal calf serum and antibiotics at 37 ◦C in a humidified atmosphere containing 5% CO2. Confluent cells in 10-cm2 petri dishes were preincubated for 4 h with or without 1 mM p-nitrophenyl β-D-xylopyranoside in MEM containing 250 µM cold Na2SO4 without FCS in 5% CO2 at 37◦C. Cells 35 were then labeled with 150 µCi/ml Na2[ S]O4 in the same medium for 24 h, as described previously [49]. At the end of the labeling period, an equal volume of 100 mM sodium acetate buffer, pH 5.8, containing 8 M urea, 4% Triton X-100, 20 mM ethylenediaminetetraacetic acid, 20 mM N-ethylmaleimide, and 1mM phenylmethanesulfonyl fluoride was added to the medium. The cell layer was harvested in 50 mM sodium acetate buffer, pH 5.8, containing 2 M urea, 2% Triton X-100, and an aliquot was used for protein content determination with the bicinchoninic acid Protein Assay (Pierce), while the remainder was added to the medium. Samples were loaded on 1 ml DEAE SephacelTM columns; after column washing with 50 mM sodium acetate buffer, pH 6.0, 8 M urea, 0.15 M NaCl, 0.5% Triton X-100 and proteinase inhibitors, PGs were eluted with 1 M NaCl in the same buffer, recovered by precipitation with 9 volumes of ethanol. The pellet was washed with 70% ethanol and then solubilized in water; PGs were quantified by measuring the 35S-activity using a liquid scintillation counter and normalized to the protein content. Labeled PGs synthesized by cells in absence of p-nitrophenyl β-D-xylopiranoside and purified as described above, were lyophilized, dissolved in 4 M guanidinium chloride, 50 mM sodium acetate buffer, pH 6.0, 0.5% Triton X-100, and analyzed by Size Exclusion Chromatography. Samples were then loaded on a Superose 6 10/300GL column (GE Healthcare) and eluted in the same buffer at 0.2 ml/min. Fractions of 0.4 ml were collected and 35S-activity was measured by scintillation counting [50]. Genes 2019, 10, 799 4 of 21

2.2. Molecular Genetic Testing Genomic DNA of each patient was isolated from peripheral blood mononuclear cells and analyzed using a capture-based IonAmpliSeq custom panel on an Ion Torrent series S5 instrument (Thermo Fisher Scientific). Results were filtered for the B4GALT7 and B3GALT6 genes. Identified variants were confirmed by PCR amplification and subsequent bidirectional Sanger sequencing, using primers encompassing the entire exon and intron–exon boundaries comprising the variants in the case of B4GALT7, or the entire coding sequence and surrounding untranslated regions in the case of B3GALT6 single exon. In parallel, DNA samples from the respective parents were analyzed by Sanger sequencing in order to evaluate parental segregation. Primer sequences are available upon request.

3. Patients and Results

3.1. First Patient

3.1.1. Clinical Report The boy is the first child of healthy, non-consanguineous parents. The pregnancy was unremarkable and the mother was not exposed to any known teratogens during pregnancy. Fetal movements were described as normal. He was born by cesarean delivery at week 38 with birth weight of 2520 g (3rd–10th centile), length 43 cm (<3rd centile), and head circumference of 32 cm (3rd–10th centile). Apgar scores were 9/10. He presented with mesomelic shortening of upper limbs, ulnar deviation of fingers and left hip dysplasia. Cerebral and renal ultrasound were normal, while echocardiography showed atrial septal defect. The baby was discharged from hospital at the age of 6 days, with diagnosis of “arthrogryposis with ulnar deviation of fingers, left hip dysplasia, and congenital heart disease”, and later he was fitted with a finger extension device and was given pelvic bimalleolar hip plaster for left hip dysplasia. He was seen at our Medical Genetics Unit at 3 months of age (Figure2): length and weight, calculated by taking into account the hip plaster, were respectively 51 cm (3rd–10th centile) and 4900 g (3rd centile), and head circumference was 41 cm (25th centile). The physical examination showed: round flat face, mild proptosis, slightly blue sclerae (Figure2A–D), joint hypermobility especially evident in the hands, mesomelic shortening of the arms, abnormal prono-supination of the left upper limbs, often flexed, short hands and feet with soft skin, bilateral ulnar deviation of fingers, 2nd finger clinodactyly, 2nd–5th finger camptodactyly on the left, and 3rd and 4th finger camptodactyly on the right (Figure2F–G). During the clinical follow-up, we noticed an improvement in the finger alterations, but a worsening of the cutaneous hyperextensibility and joint hypermobility. At the age of 3 years and 7 months, he presented with round face, blue sclerae, proptosis (Figure2D), soft hyperextensible skin and extreme joint hypermobility (Beighton score: 5) in particular of hands and feet, with significant improvement in finger ulnar deviation, clinodactyly, camptodactyly and overlapping toes (Figure2H–K). Height and weight were 87 cm (<3rd centile) and 9920 g (<3rd centile), head circumference was 49 cm (10th centile). Span was 82 cm and span/height ratio was 0.94. Early psychomotor development was delayed at least in part owing to his orthopedic conditions. He gained head control at 3 months, was able to sit at 3 years and walk aided at the age of 4 years. He uttered his first words at 2 years, but his language skills improved rapidly and significantly thereafter: at 5 years, psychological evaluation reported an IQ of 105. The skeletal X-rays demonstrated wide anterior ribs, bilateral bowed ulna and radius with dislocation/subluxation and radioulnar synostosis, bilateral metaphyseal widening of the radius, bilateral short and dysmorphic 2nd finger middle phalanx, short first metacarpal (Figure3A–F). Genes 2019, 10, 799 5 of 21 Genes 2019, 10, 799 5 of 23

Figure 2. PatientPatient 1 1AA) Age) Age 3 months: 3 months: ulnar ulnar deviation deviation of fingers; of fingers; B) AgeB) Age 3 months: 3 months: round round flat face, flat mild face, mildproptosis, proptosis, mesomelic mesomelic shortening shortening of upper of upperlimbs, limbs,folded foldedskin on skin forearm; on forearm; C) AgeC 3) months: Age 3 months: flat profile; flat profile;D, F–GD,) Age F–G )3 Agemonths: 3 months: ulnar ulnar deviation deviation of fingers, of fingers, 2nd 2nd finger finger clinodac clinodactyly,tyly, 2nd–5th 2nd–5th fingerfinger camptodactyly on thethe leftleft hand,hand, 3rd3rd andand 4th4th fingerfinger camptodactyly camptodactyly on on the the right right hand; hand;E )E Age) Age 3 3 years years 7 months:7 months: round round face, face, blue blue sclerae, sclerae, proptosis; proptosis;H– HK)–K Age) Age 3 years 3 years 7 months: 7 months: soft, soft, hyperextensible hyperextensible skin skin and extreme joint hypermobility (Beighton score: 5) in particular of hands and feet; significant improvement Genes 2019and, 10 extreme, 799 joint hypermobility (Beighton score: 5) in particular of hands and feet; significant 6 of 23 inimprovement finger ulnar in deviation, finger ulnar clinodactyly, deviation, camptodactyly clinodactyly, of thecamptodactyly second finger, of and the overlapping second finger, toes. and overlapping toes.

During the clinical follow-up, we noticed an improvement in the finger alterations, but a worsening of the cutaneous hyperextensibility and joint hypermobility. At the age of 3 years and 7 months, he presented with round face, blue sclerae, proptosis (Figure 2 D), soft hyperextensible skin and extreme joint hypermobility (Beighton score: 5) in particular of hands and feet, with significant improvement in finger ulnar deviation, clinodactyly, camptodactyly and overlapping toes (Figure 2 H–K). Height and weight were 87 cm (<3rd centile) and 9920 g (<3rd centile), head circumference was 49 cm (10th centile). Span was 82 cm and span/height ratio was 0.94. Early psychomotor development was delayed at least in part owing to his orthopedic conditions. He gained head control at 3 months, was able to sit at 3 years and walk aided at the age of 4 years. He uttered his first words at 2 years, but his language skills improved rapidly and significantly thereafter: at 5 years, psychological evaluation reported an IQ of 105. The skeletal X-rays demonstrated wide anterior ribs, bilateral bowed ulna and radius with dislocation/subluxation and radioulnar synostosis, bilateral metaphyseal widening of the radius, bilateralFigureFigure short 3. 3.PatientPatient and dysmorphic1 1AA–B–B, ,DD––EE) )Bilateral2nd Bilateral finger bowing bowing middle of ulnaphalanx,ulna andand radiusradiusshort withfirst with metacarpal dislocation dislocation/subluxation/subluxation (Figure 3 A–F). and and radioulnar synostosis, metaphyseal widening of the radius; C) bilateral short and dysmorphic 2nd radioulnar synostosis, metaphyseal widening of the radius; C) bilateral short and dysmorphic 2nd finger middle phalanx, short first metacarpal; F) hip dysplasia. finger middle phalanx, short first metacarpal; F) hip dysplasia. Electrencephalography, electromyography and brain magnetic resonance imaging were all normal. AtElectrencephalography, the age of 5 years, molecular electromyography analysis of B4GALT7 and wasbrain performed, magnetic showingresonance the imaging presence ofwere two all normal.novel pathogenicAt the age of mutations, 5 years, indicativemolecular ofanalysis spEDS. of B4GALT7 was performed, showing the presence of two novel pathogenic mutations, indicative of spEDS. 3.1.2. Molecular and Functional Analysis 3.1.2. Molecular and Functional Analysis Karyotype was normal, 46,XY. Our first diagnostic hypothesis was either myopathy or collagen KaryotypeVI pathology, was normal, but molecular 46,XY. analysisOur first of diagnostic the COL6A1 hypothesis, COL6A2 was, COLA613 either ,myopathyRAPSN genes or collagen did not VI pathology, but molecular analysis of the COL6A1, COL6A2, COLA613, RAPSN genes did not reveal any rare or pathogenic variant. Subsequently, by analyzing a panel of EDS-related genes, two variants were identified in compound heterozygosity in exons 2 and 3 of the B4GALT7 gene:

NM_007255:c.[277dupC];[628C>T], NP_009186:p.[(His93Profs*73)];[(His210Tyr)] (Figure 4).

Figure 4. A) Pedigree of Family 1; B) Schematic representation of the B4GALT7 protein: TM = transmembrane domain, GalT = Galactosyl-transferase catalytic domain. Protein variants reported in the literature are annotated in the cartoon (see Table 1 for references). Top half: missense variants (black). Bottom half: truncating variants (red).

Genes 2019, 10, 799 6 of 23

Figure 3. Patient 1 A–B, D–E) Bilateral bowing of ulna and radius with dislocation/subluxation and radioulnar synostosis, metaphyseal widening of the radius; C) bilateral short and dysmorphic 2nd finger middle phalanx, short first metacarpal; F) hip dysplasia.

Electrencephalography, electromyography and brain magnetic resonance imaging were all normal. At the age of 5 years, molecular analysis of B4GALT7 was performed, showing the presence of two novel pathogenic mutations, indicative of spEDS.

3.1.2. Molecular and Functional Analysis Genes 2019, 10, 799 6 of 21 Karyotype was normal, 46,XY. Our first diagnostic hypothesis was either myopathy or collagen VI pathology, but molecular analysis of the COL6A1, COL6A2, COLA613, RAPSN genes did not reveal anyreveal rare any or pathogenic rare or pathogenic variant. Subsequently, variant. Subsequently, by analyzing by a analyzing panel of EDS-related a panel of genes, EDS-related two variants genes, weretwo variants identified were in identiﬁed compound in compound heterozygosity heterozygosity in exons in 2 exons and 2 and3 of 3 ofthe the B4GALT7B4GALT7 gene:gene: NM_007255:c.[277dupC];[628C>T],NM_007255:c.[277dupC];[628C>T], NP_009186:p.[( NP_009186:p.[(His93Profs*73)];[(His210Tyr)]His93Profs*73)];[(His210Tyr)] (Figure4 4).).

Figure 4. A A)) Pedigree Pedigree of of Family 1; B) SchematicSchematic representationrepresentation ofof thethe B4GALT7B4GALT7 protein:protein: TMTM == transmembrane domain, domain, GalT GalT == Galactosyl-transferaseGalactosyl-transferase catalytic catalytic domain. domain. Protein Protein variants variants reported reported in thein the literature literature are are annotated annotated in inthe the cartoon cartoon (see (see Table Table 1 for references).references). TopTop half:half: missensemissense variantsvariants (black). Bottom Bottom half: truncating truncating variants variants (red). (red).

The single nucleotide insertion c.277dupC, inherited from the mother, creates a new reading frame, terminating with a premature stop codon 73 residues downstream, and its transcript is predicted to undergo nonsense-mediated decay (https://nmdprediction.shinyapps.io/nmdescpredictor/). This variant was also described in another patient by Salter et al. in 2016 [32]. The heterozygous C>T transition at nucleotide 628, inherited from the father, results in a non-conservative Histidine to Tyrosine substitution at codon 210 and occurs in a highly conserved residue. This variant has not been reported before, but it is considered deleterious by multiple prediction algorithms (MutationTaster, PolyPhen2, SIFT) and is absent from reference population databases (gnomAD, 1000 Genomes). Based on these criteria and on the clinical features of the patient, we assume that the combination of these variants in trans is causative. The study of PG synthesis on patient’s fibroblasts obtained by skin biopsy did not reveal any significant differences compared to normal controls, neither at basal conditions nor in the presence of beta-xyloside to enhance glycosaminoglycan synthesis. However, the level of PG synthesis in the patient’s fibroblasts was at the lower normal limit. Analysis through gel filtration to assess the fraction of whole PGs with respect to glycosaminoglycans did not show any differences. Although these results were not significant per se, the slightly reduced PG levels combined with the genetic testing offered some indication that the variants identified in B4GALT7 may in fact have a deleterious effect on protein function. Genes 2019, 10, 799 7 of 21

Table 1. Patients with B4GALT7 variants: Review of the literature.

Genetics [808C>T] heterozygous p.[(H93Pfs*73)]; Variants on p.[(A186D)]; p.[(R270C)]; p.[(L41P)]; p.[(R270C)]; p.[(C324S)]; [(C214Y)] p.[(E277*)]; p.[(R141W)]; p.[(H93Pfs*73)]; ? protein [(L206P)] [(R270C)] [(R270C)] [(R270C)] [(C324S)] p.[(R141W)]; [(E277*)] [(R270C)] [(H210Y)] [(R270C)] Gender 5M M 1F 1M M 11M 11F F 1M 1F F 1M 1F M 17M 16F 8y, 15y, 15y, 16y, Age 4y 9m 2y, 33y 10y 4y, 46y 5y 3y 6m, 13y 30y 4y, 10y 7y 8m 2y 46y 18y → Short staturea 4/5 + 2/2 + 22/22 + 2/2 + 2/2 + 33/33 Radiolunar n.a. + 2/2 + 10/21 - 2/2 - 2/2 + 19/32 synostosisc Bowing of n.a. + 2/2 + 21/21 - 2/2 - 2/2 + 30/32 limbsa Joint hypermobility 5/5 + 2/2 + 22/22 + 2/2 + 2/2 + 33/33 especially of the Main features handsc Skin hyperextensibility, 5/5 + 2/2 + 21/22 + 2/2 + 2/2 + 32/33 soft, doughy skinb Progeroid facial 5/5 mild 0/2 - 0/22 - 0/2 - 0/2 - 1/33 appearance c Short face - + 2/2 - + 2/2 - 2/2 + 31/33 Midface - - 2/2 - + 2/2 - 2/2 + 30/33 hypoplasia 22/22 Narrow mouth - + 2/2 - + 1/2 + 2/2 + 31/33 Proptosis - + 2/2 - + 2/2 + 2/2 + 32/33

Facial dysmorphisms Cleft palate - Biﬁd uvula 0/2 - 1/22 - 1/2 - 1/2 - 4/33 Loose skin - + 2/2 + n.a. n.a. 2/2 + n.a. + 8/8 Genes 2019, 10, 799 8 of 21

Table 1. Cont.

Hernandez et al. Kresse et al. Faiyaz-Ul-Haque Guo et al. Cartault et al. Arunrut et al. Salter et al. Ritelli et al. Sandler-Wilson This study, Total (patients [1979, 1981, 1986] [1987][24] et al. [2004][29] [2013][30] [2015][31] [2016][33] [2016][32] [2017][51] et al. [2019][52] Patient 1 with mutation) [21–23] Delayed wound 5/5 + 1/2 + n.a. + 2/2 + n.a - 7/8 healing 1/5 Cardiovascular Aortic/Pulmonic n.a. n.a. - n.a. - n.a. - n.a. + :ASD 1/4 abnormalities Stenosis Delayed motor 5/5 + 2/2 - n.a. + 2/2 + 2/2 + 10/11 developmentb Delayed Mild learning 12/22 (learning 1/2 Severe cognitive 5/5 n.a. n.a. n.a. - 2/2 - 16/29 disabilities disabilities) 1/2 n.a developmentb Muscle n.a. + 2/2 mild n.a. + 2/2 + 2/2 + 11/11 hypotoniaa Nystagmus Iris and optic nerve Severe 1/2 Mild Severe hyperopia, colobomas hypermetropia 5/21 glaucoma 2/2 Blue sclerae Ophthalmological esotropia and congenital ptosis, Posterior Small optic n.a. - 1/21 - 1/2 Severe Myopia 13/32 abnormalitiesc mild intermittent subcapsular nerves megalocornea hyperopia hypermetropia exotropia cataracts Hypermetropia High hyperopia Strabismus Right-sided ptosis Osteopeniab n.a. + 2/2 - n.a. n.a. 2/2 + 1/2 + 8/10 1/2 Pes planusb 5/5 + 1 + n.a. + + 2/2 + 9/11 2 1/2 n.a. Other clinical features Bilateral elbow contractures or n.a. + 2/2 + n.a. n.a. 2/2 - 1/2 + 8/10 limited elbow movementc Sensorineural 1/2 Conductive n.a. - n.a. n.a. n.a. n.a. + n.a. - 2/7 hearing loss hearing loss Pectus Cryptorchidism Yellow carinatum 1/2 Chest wall 4/5 discoloration of Scoliosis deformity Inguinal hernia teeth with Pectus Broad fingertips Irregular and Bilateral hallux Dental anomalies: 2/2 Coronal 1/5 defective enamel carinatum 5/22 Subluxation of fragile dentition valgus Other less defective and cleft vertebrae Hip dysplasia Hypogonadism Mild eventration Bifid thumb the distal Scoliosis Lymphedema frequent greyish enamel Unilateral ptosis 2/2 Sagittal Ulnar deviation 1/5 of the right 2/22 interphalangeal Bilateral Scoliosis features Clavicular craniosynostosis of fingers Varicose veins 5/5 hemidiaphragm Scoliosis/ joints patellar Temporomandibular exostoses 1/2 Multiple nevi 5/5 Bilateral Kyphosis 6/22 Absence of the dislocation joint dislocation Vesicoureteral Dental anomalies equinovarus pineal gland reflux 5/5 deformity Prominent scalp veins EDS: Ehlers–Danlos syndrome; n.a. not available; ASD: atrial septal defect; aSpondylodysplastic EDS (spEDS) major criteria [46]; bspEDS minor criteria [46]; cspEDS specific minor criteria for B4GALT7 variants [46]. The 5 patients from Hernandez et al. (column 1) had clinical data compatible with spEDS-B4GALT7, but were not considered in the clinical signs totals because it was unclear whether the clinical diagnosis was confirmed by molecular testing or not [21–23]. Genes 2019, 10, 799 9 of 21

3.2. Second Patient

3.2.1. Clinical Report This patient was partially described in Nakajima et al., 2013 as P9 [45]. He is the first child of healthy non-consanguineous parents, born at the 40th week of gestation by vaginal delivery. During pregnancy, no known exposures to potential teratogens were detected. The mother reported normal fetal movements. Prenatal ultrasound showed bilateral megaureter. At birth, weight was 2870 g (10th centile), length was 48 cm (3rd–10th centile) and head circumference measured 34 cm (25th centile). He presented with: joint hypermobility, soft skin, bilateral congenital talipes equinovarus and right hip dysplasia, which was treated with a Pavlik harness. Renal ultrasound showed a mild bilateral caliceal dilatation, while renal scintigraphy was normal. During the first year of life he was diagnosed with early-onset scoliosis, and he began to wear an orthopedic corset from the age of 4 years. Two years later. he was operated for scoliosis after correction with Halo Gravity Traction and he subsequently received three operations for the lengthening of the pins (until the age of 10 years). He suffered from frequent fractures: a fracture of the left femur after a low trauma at the age of 5 years and 6 months and to the right femur at the age of 8 years and 8 months and a micro-fracture of the right proximal fibula in an area of reduced bone density, compatible with an osseous cyst, at the age of 10 years. Furthermore, he had recurrent luxation of the toes. Early psychomotor development was normal, but he later suffered from motor delay due to his skeletal and large joint anomalies. He gained head control at the age of 2 months and could sit up at the age of 8 months. He could walk unaided at the age of 2 years and uttered his first words at the age of 1 year. He currently attends the first year of middle school with a support teacher owing to a diminished ability to concentrate. He does not present with intellectual disability or other behavioral disturbances. At the age of 12 years and 7 months (Figure5), height was 109cm ( <3rd centile <-6SD), weight 29 kg (<3rd centile), head circumference 54 cm (50th–75th centile), span 114 cm, span/height ratio 1.06. Pubertal stage was A0P1B1, with testicular volume 1–2 ml on the left side and 2–3ml on the right. He had sparse hair, high and prominent forehead, large ears, sparse eyebrows, large deeply-set eyes, blue-gray sclerae, hypoplastic columella and misalignment of teeth, which were small, with abnormal enamel and yellow-brown discoloration (Figure5A–C). He had thin, pale, extremely soft skin, with a prominent venous patterning on the trunk and limbs, limited elbow extension, skin hyperextensibility and distal joint hypermobility especially of the hands (Beighton scale: 6), long and tapered fingers, normal nails. His feet had hypoplastic nails, short and overlapping toes, hallux valgus (Figure5D–H). The skeletal X-rays demonstrated severe kyphoscoliosis, osteopenia, thin metacarpals and phalanges (Figure6A–H). Audiometric and ophthalmological evaluations were normal. Abdominal ultrasound showed ptosic kidneys, bilateral pelvic ureteral dilatation and thickening of the bladder walls. Echocardiography demonstrated mild dilatation of the aortic bulb and mild mitral valve prolapse. Genes 2019, 10, 799 11 of 23 tapered fingers, normal nails. His feet had hypoplastic nails, short and overlapping toes, hallux valgus (Figure 5 D–H). Genes 2019, 10, 799 11 of 23

Genestapered2019 ,fingers,10, 799 normal nails. His feet had hypoplastic nails, short and overlapping toes, hallux10 of 21 valgus (Figure 5 D–H).

Figure 5. Patient 2 A–C) Sparse hair, high and prominent forehead, sparse eyebrows, deeply-set eyes, blue sclerae, hypoplastic columella, large ears; D–H) Thin, pale, extremely soft skin, with prominent veins on the trunk and limbs, limited elbow extension, skin hyperextensibility and distal joint hypermobility especially of the hands, long and tapered fingers. Feet: hypoplastic nails, short, Figure 5. PatientPatient 2 2 AA––CC)) Sparse Sparse hair, hair, high high and and prominent prominent forehe forehead,ad, sparse sparse eyebrows, eyebrows, deeply-set deeply-set eyes, eyes, overlapping toes, hallux valgus. blue sclerae, hypoplastic columella, large ears; D–H) Thin, pale, extremely softsoft skin, with prominent veins on the trunk and limbs, limited elbow extension, skin hyperextensibility and distal joint Theveins skeletalon the trunkX-rays and demonstrated limbs, limited severeelbow extenskyphoscoliosis,ion, skin hyperextensibility osteopenia, thin and metacarpals distal joint and hypermobility especially of the hands,hands, longlong andand taperedtapered ﬁngers.fingers. Feet: hypoplastic hypoplastic nails, short, phalanges (Figure 6 A–H). overlapping toes, hallux valgus.

The skeletal X-rays demonstrated severe kyphoscoliosis, osteopenia, thin metacarpals and phalanges (Figure 6 A–H).

FigureFigure 6. Patient 6. Patient 2 A– 2H A) Severe,–H) Severe, early early onset onset kyphoscoliosis, kyphoscoliosis, osteopenia, osteopenia, thin metacarpals thin metacarpals and phalanges. and phalanges. 3.2.2. Molecular Analysis

AudiometricSanger sequencing and ophthalmologic confirmed theal two evaluations previously were reported normal. variants Abdominal in the ultrasound single exon showed of the Figure 6. Patient 2 A–H) Severe, early onset kyphoscoliosis, osteopenia, thin metacarpals and B3GALT6ptosic kidneys,gene [45 ]:bilateral NM_080605:c.[353delA];[925T pelvic ureteral dilatation>A], NP_542172:p.[(Asp118Alafs*160)];[(Ser309Thr)] and thickening of the bladder walls. phalanges. (FigureEchocardiography7A,B). demonstrated mild dilatation of the aortic bulb and mild mitral valve prolapse. TheAudiometric frameshift and variant ophthalmologic was inheritedal from evaluations the father were and thenormal. missense Abdominal variant from ultrasound the mother. showed Both 3.2.2. Molecular Analysis variantsptosic kidneys, can be considered bilateral pathogenic,pelvic ureteral according dilatation to the predicted and thickening effect on the of protein, the bladder the consistency walls. ofEchocardiography theSanger clinical sequencing phenotypes, demonstrated confirmed and theirmild the dilatation raritytwo previously in referenceof the aortic reported population bulb variants and mild databases in mitral the single such valve asexon prolapse. gnomAD of the (MAFB3GALT6=0.0026% and absent,generespectively). Variant[45]: c.353delA hasNM_080605:c.[353delA];[925T>A], also been found in our third patientNP_542172:p.[(Asp118Alafs*160)];[(S3.2.2. Molecular (reported Analysis below), while varianter309Thr)] c.925T> A(Figure had already 7A,B). been reported in other patients in the literature [45,48]. Since this gene is encoded by a single exon, its frameshift variants, identified in various patients,Sanger are sequencing expected to escapeconfirmed nonsense-mediated the two previously mRNA reported decay. variants They probably in the single exert a exon pathogenic of the eB3GALT6ffect byleading to an unstablegene transcript or[45]: an unstable /non-functionalNM_080605:c.[353delA];[925T>A], protein, as suggested by experimentalNP_542172:p.[(Asp118Alafs*160)];[(S evidence [48]. er309Thr)] (Figure 7A,B).

Genes 2019, 10, 799 11 of 21 Genes 2019, 10, 799 12 of 23

Figure 7. A A)) Pedigree Pedigree of of Families Families 2 2 and and 3; 3; B)) Schematic Schematic representation of the B3GALT6 protein:protein: TM = transmembranetransmembrane domain, domain, GalT GalT == Galactosyl-transferaseGalactosyl-transferase catalytic catalytic domain. domain. Protein Protein variants variants reported reported in thein the literature literature are are annotated annotated in inthe the cartoon cartoon (see (see Table Table 22 for references). Top half:half: missensemissense variantsvariants (black). Bottom Bottom half: half: frameshift frameshift variants variants (red), (red), in-fr in-frameame indels indels (green), (green), other length-altering length-altering variants variants (blue); C) Protein Protein projection of the two alleles from from Pt.3 Pt.3;; dashed dashed boxes boxes indicate indicate an an altered altered or or extended extended readingreading frame. 3.3. Third Patient The frameshift variant was inherited from the father and the missense variant from the mother. Both3.3.1. variants Clinical Reportcan be considered pathogenic, according to the predicted effect on the protein, the consistency of the clinical phenotypes, and their rarity in reference population databases such as He is the second child of non-consanguineous parents, born at 35th week of gestation by caesarean gnomAD (MAF=0.0026% and absent, respectively). Variant c.353delA has also been found in our section, owing to the premature rupture of the amniotic sac. During pregnancy, no known exposures third patient (reported below), while variant c.925T>A had already been reported in other patients in to potential teratogens were detected. Prenatal ultrasound indicated bilateral talipes equinovarus. the literature [45,48]. Since this gene is encoded by a single exon, its frameshift variants, identified in The mother reported normal fetal movements. At birth, weight was 2500 g (50th centile), length was various patients, are expected to escape nonsense-mediated mRNA decay. They probably exert a 47 cm (50th–75th centile) and head circumference measured 34 cm (90th centile). Apgar scores pathogenic effect by leading to an unstable transcript or an unstable/non-functional protein, as were 1’:7 5’:9. He presented with joint hypermobility, soft skin, adducted thumbs, right talipes suggested by experimental evidence [48]. equinovarus. In the second year of life he was diagnosed with early-onset scoliosis, and he began to wear an orthopedic corset. 3.3. Third Patient At age 6 years, he was operated for bilateral cryptorchidism and at age 9 years, he underwent 3.3.1.a gastrostomy Clinical report to correct Barrett’s oesophagus. His motor development was delayed. He gained head control at the age of 4–5 months and could sit upHe at theis the age second of 24 months. child of He non-consanguineous could walk aided at theparents, age of born 5 years, at he35th was week able of to walkgestation alone by at caesareanthe age of section, 6 years andowing 6 months to the premature and uttered rupture his ﬁrst ofwords the amniotic at the agesac. of During 1 year. pregnancy, He currently no attendsknown exposuresthe second to year potential of middle teratogens school with were a support detected. teacher Prenatal owing ultrasound to a diminished indicated ability bilateral to concentrate. talipes equinovarus.He does not present The mother with intellectual reported normal disability fetal or othermovements. behavioral At disturbances.birth, weight was 2500 g (50th centile),Examination length was at 47 the cm age (50th–75th of 12 years centile) and 11 and months head circumference showed (Figure measur8A–F):ed height 34 cm 123 (90th cm centile). ( <<3rd Apgarcentile), scores weight were 28 kg1’:7 (< 5’:9.3rd He centile), presented head circumferencewith joint hypermobility, 52 cm (10th soft centile), skin, span adducted 113 cm, thumbs, span/height right talipesratio 0.91. equinovarus. Pubertal stage In the were second A0P1B1, year of with life testicular he was diagnosed volume of with 1-2 mlearly-onset bilaterally. scoliosis, He had and sparse he beganhair, high to wear and an prominent orthopedic forehead corset. with high hairline, mild bitemporal depression, fairly large ears, blue-grayAt age sclerae, 6 years, malar he was hypoplasia, operated hypoplasticfor bilateral columella,cryptorchidism short and philtrum, at age small 9 years, misaligned he underwent teeth ina gastrostomythe lower jaw, to ﬂatcorrect palate Barrett’s with longitudinal oesophagus. median mucous thickening (Figure8A–B). He had thin, His motor development was delayed. He gained head control at the age of 4–5 months and could sit up at the age of 24 months. He could walk aided at the age of 5 years, he was able to walk alone

Genes 2019, 10, 799 13 of 23 at the age of 6 years and 6 months and uttered his first words at the age of 1 year. He currently attends the second year of middle school with a support teacher owing to a diminished ability to concentrate. He does not present with intellectual disability or other behavioral disturbances. Examination at the age of 12 years and 11 months showed (Figure 8 A–F): height 123 cm (<<3rd centile), weight 28 kg (<3rd centile), head circumference 52 cm (10th centile), span 113 cm, span/height ratio 0.91. Pubertal stage were A0P1B1, with testicular volume of 1-2 ml bilaterally. He had sparse hair, high and prominent forehead with high hairline, mild bitemporal depression, fairly large ears, blue-gray sclerae, malar hypoplasia, hypoplastic columella, short philtrum, small misaligned teeth in Genes 2019, 10, 799 12 of 21 the lower jaw, flat palate with longitudinal median mucous thickening (Figure 8 A–B). He had thin, pale, soft skin, with slightly prominent veins on trunk, limited elbow extension, skin pale,hyperextensibility soft skin, with and slightly distal prominent joint hypermobility veins on trunk, especially limited of elbow the hands extension, (Beighton skin scale: hyperextensibility 6), long and taperedand distal fingers joint with hypermobility a tendency especiallyto ulnar deviation of the hands (Figure (Beighton 8 C–E), hypoplastic scale: 6), long nails and on taperedthe feet (Figure ﬁngers with8 F). a tendency to ulnar deviation (Figure8C–E), hypoplastic nails on the feet (Figure8F).

Figure 8. PatientPatient 3 3 AA,,BB)) Sparse Sparse hair, hair, high high and and prominent prominent forehead forehead with with high high hairline, hairline, mild mild bi-temporal bi-temporal depression, fairlyfairly large large ears, ears, blue-gray blue-gray sclerae, sclerae malar, malar hypoplasia, hypoplasia, hypoplastic hypoplastic columella, columella, short philtrum, short philtrum,thin, pale, thin, soft skinpale, with soft prominentskin with prominent veins on the veins trunk; on Cthe–E )trunk; Skin hyperextensibilityC–E) Skin hyperextensibility and distal jointand distalhypermobility joint hypermobility especially of especially the hands, of longthe hands, and tapered long and ﬁngers, tapered with fingers, a tendency with to a ulnartendency deviation; to ulnarF) deviation;Feet: hypoplastic F) Feet:nails. hypoplastic nails.

Skeletal X-rays demonstrated se severevere kyphoscoliosis, osteopenia osteopenia (especially (especially in in the the acetabula, femurs,Genes 2019 tibiae, 10, 799 and ﬁbulae),fibulae), and thin metacarpals,metacarpals, metatarsalsmetatarsals andand phalangesphalanges (Figure(Figure9 9A–F). A–F). 14 of 23

Figure 9. Patient 3 A–F) Severe early onsetonset kyphoscoliosis;kyphoscoliosis; osteopeniaosteopenia of the acetabula, femur, tibiae and ﬁbulae;fibulae; thinthin metacarpals,metacarpals, metatarsalsmetatarsals andand phalanges.phalanges.

Audiometric andand ophthalmological ophthalmologic evaluationsal evaluations were were normal. normal. Abdominal Abdominal ultrasound ultrasound was normal. was Echocardiographynormal. Echocardiography demonstrated demonstrated an atrial septalan atrial defect septal and defect mild mitralicand mild insu mitralicﬃciency insufficiency with thickened with mitralicthickened valve. mitralic valve.

3.3.2. Molecular Analysis Karyotype was normal, 46XY. Genetic testing revealed three variants in the B3GALT6 gene: NM_080605:c.[308C>T;353delA];[987_989delCTG], NP_542172:p.[(Ala103Val);(Asp118Alafs*160)];[(*330Alaext*73)] (Figure 7 A–C). Variant c.353delA, already described as pathogenic (patient 2 and Nakajima et al., 2013 [45]), has been found in cis with variant c.308C>T, both inherited from the healthy father. This missense variant is absent from the reference population databases (gnomAD, 1000 genomes), but occurs at a poorly conserved position and is predicted as benign by multiple algorithms (MutationTaster, PolyPhen2, SIFT). It should be classified as a variant of uncertain significance, as there is not enough evidence to suggest whether this variant alone would be tolerated or damaging to protein function, or to indicate a possible contribution to the deleterious effect of the frameshift variant. Variant c.987_989delCTG, inherited from the healthy mother, disrupts the constitutive stop codon leading to an extended open reading frame, encoding a 72aa longer protein. It is absent from reference population databases (gnomAD, 1000 genomes), and by analogy with the frameshift variants in this gene, it is expected to produce an unstable or non-functional protein. Since it was found in trans with a recognized deleterious mutation, this variant can be considered as likely pathogenic.

Genes 2019, 10, 799 13 of 21

Table 2. Patients with B3GALT6 variants: Review of the literature.

Malfait et al. Nakajima et al. Sellars et al. Ritelli et al., Alazami et al., Trejo Ben-Mahmoud Van Damme et al. This study, This study, Total (patients 2013[44] 2013[45] 2014[53] 2015[54] 2016[18] et al.2017[55] et al. 2018[56] 2018[48] Patient 2 Patient 3 with mutation) B3GALT6 EDS-like EDS-like Diagnosis spectrum EDS progeroid syndrome spEDS- connective tissue B3GALT6- (Various SEMDJL1 type2 EDS progeroid SEMDJL1 B3GALT6 spEDS- B3GALT6 spEDS- B3GALT6 spEDS- B3GALT6 spEDS- B3GALT6 disorder phenotype denominations) EDS-progeroid SEMDJL1 type2 SEMDJL1 SEMDJL1 form SEMDJL1 c.[619G>C]; 9 Compound [619G>C] c.[556T>C]; 8 Compound 12/45 homozygous heterozygous, B3GALT6 c.[323_344del]; c.[227delT]; [556T>C] c.[511C>T]; c.[618C>G]; heterozygous, c.[353delA]; c.[308C>T;353delA]; 32/45 compound 1 Heterozygous; ? variants [619G>C] [766C>T] c.[536_541dup]; [901_921dup] [618C>G] 1 Homozygous; [925T>A] [987_989delCTG] heterozygous see Ref. (Table c.[649G>A]; [536_541dup] see Ref (Table 3) 1/45 heterozygous Genetics 3) [649G>A] p.[(D207H)]; [(D207H)] p.[(F186L)]; p.[(A103V); Variants on p.[(A108Gfs*163)]; p.[(D159Y)]; p.[(I76Tfs*202)]; [(F186L)] p.[(R171C)]; p.[(C206W)]; p.[(D118Afs*160)]; See Figure7 See Figure7 (D118Afs*160)]; protein [(D207H)] [(E265D)] [(R256W)] p.[(R179_R180dup)]; [(K301_R307dup)] [(C206W)] [(S309T)] [(*330Aext*73)] p.[(G217S)]; [(R179_R180dup)] [(G217S)] Gender 2M 3F 6M 6F M 2F 1M 4F 3F 1M 2F 7M 5F M M 20M 25F Age 1y8m 27y 1m 34y 6m 21y, 25y 6w 6y 12y, 15y, 15y 4d 2m 8m 37y 12y 7m 13y 3m 4d 37y → → → → → → Short stature a 2/3 12/12 + 2/2 3/3 3/3 n.a. 10/10 + + 35/36 Kyphoscoliosis (congenital or 3/4 12/12 - 2/2 4/5 3/3 n.a. 10/10 + + 36/39 early onset, progressive) c Bowing of limbs a 2/4 n.a. + n.a. n.a. 3/3 n.a. 10/10 - - 16/20 Joint hypermobility 4/4 7/10 n.a. 2/2 5/5 3/3 2/2 10/10 + + 33/36 especially of the hands c Main features Skin hyperextensibility, 4/4 6/10 n.a. 2/2 4/5 3/3 2/2 9/10 + + 33/38 soft, doughy skin b

Skeletal changes 3/4 12/12 n.a. 2/2 2/2 0/1 3/3 10/10 + + 34/36 SEMDJL1 d Prominent c 4/4 9/10 + 2/2 4/5 0/3 1/1 8/10 + + 31/38 forehead Sparse hair 2/4 3/10 - 0/2 n.a 2/3 0/1 3/3 + + 12/26 Midface 2/4 n.a. + 1/2 4/5 2/3 1/1 8/10 + + 21/28 hypoplasia Blue sclerae 3/4 7/10 n.a. 2/2 4/5 1/3 n.a. 6/10 + + 25/36 Proptosis 2/4 7/10 + 0/2 n.a. 3/3 n.a. 7/10 - - 20/32 Facial dysmorphisms Cleft palate 0/4 1/10 - 0/2 n.a. 0/3 - - - - 2/25 Genes 2019, 10, 799 14 of 21

Table 2. Cont.

Other clinical features 3/4 n.a. n.a. 2/2 n.a. n.a n.a. 8/9 + + 13/17 discoloration of teeth c Sensorineural and conductive hearing Elbow loss 1/10 Prominent dislocation 9/10 Cervical spine superficial veins Limited elbow 3/3 Bilateral Contractures of instability 3/7 Limited elbow movement 9/11 radioulnar the large joints Prominent Laryngeal cleft 1/10 extension Excessive Carpal 4/5 Multiple dislocation 2/2 superficial veins Tracheomalacia 2/10 Ptosic kidney, wrinkling of synostosis 1/10 fractures 3/3 Hip Radioulnar Barrett’s Spontaneous repeated Bilateral caliceal palmar skin Short Radioulnar 2/2 Genu valgus Bilateral dysplasia synostosis 2/2 oesophagus Less frequent pneumothoraces 1/10 and ureteral (hands and feet) metacarpals synostosis 2/2 Hallux disclocated radial 2/3 Hearing loss Oligodactyly of Limited elbow features Chronic respiratory dilatation 2/4 6/10 Early death valgus head 1/3 Pectus the right 3rd extension insufficiency 2/10 Fractures Pectus deformity Hip dislocation 1/5 Pectus carinatum finger 1/2 Bilateral Pectus carinatum 1/10 Right hip dysplasia 3/4 5/12 carinatum 1/3 Ulnar Spontaneous cryptorchidism excavatum 1/10 Recurrent luxation Epiphyseal deviation of the fractures 2/2 Hypoplastic nails Wilms tumor 1/10 of the toes, dysplasia of fingers Early death 3/3 Joint dislocations 10/10 Hypoplastic nails femoral head Hip dysplasia 4/6 Hallux valgus 4/12 Fractures 8/9 Hallux valgus 3/10 EDS: Ehlers Danlos syndrome; n.a. not available; ASD: atrial septal defect; aSpondylodysplastic EDS (spEDS) major criteria [46]; bspEDS minor criteria [46]; cspEDS specific minor criteria for B3GALT6 variants[46]; d Platyspondyly, short ilia, elbow malalignment. Cases from 10 additional families reported by Vorster et al. in 2015 [42] were not listed here due to difficult data interpretation; they include an additional variant, c.235A>C, p.(T79A). Genes 2019, 10, 799 15 of 21

3.3.2. Molecular Analysis Karyotype was normal, 46XY. Genetic testing revealed three variants in the B3GALT6 gene: NM_080605:c.[308C>T;353delA];[987_989delCTG], NP_542172:p.[(Ala103Val);(Asp118Alafs*160)]; [(*330Alaext*73)] (Figure7A–C). Variant c.353delA, already described as pathogenic (patient 2 and Nakajima et al., 2013 [45]), has been found in cis with variant c.308C>T, both inherited from the healthy father. This missense variant is absent from the reference population databases (gnomAD, 1000 genomes), but occurs at a poorly conserved position and is predicted as benign by multiple algorithms (MutationTaster, PolyPhen2, SIFT). It should be classified as a variant of uncertain significance, as there is not enough evidence to suggest whether this variant alone would be tolerated or damaging to protein function, or to indicate a possible contribution to the deleterious effect of the frameshift variant. Variant c.987_989delCTG, inherited from the healthy mother, disrupts the constitutive stop codon leading to an extended open reading frame, encoding a 72aa longer protein. It is absent from reference population databases (gnomAD, 1000 genomes), and by analogy with the frameshift variants in this gene, it is expected to produce an unstable or non-functional protein. Since it was found in trans with a recognized deleterious mutation, this variant can be considered as likely pathogenic.

4. Discussion Biallelic variants in the B4GALT7 and B3GALT6 genes are responsible for conditions characterized by a combined skeletal and connective tissue phenotype. Their recent classification as spEDS [46] provides a valid assistance for their diagnosis, but the observation of new patients and the study of the clinical signs of those already reported in the literature can further improve our knowledge. In an effort to better define both the similarities and the peculiarities of these syndromes, we reviewed the notable features of our three patients and compared them to those of molecularly confirmed B4GALT7- and B3GALT6-related cases reported in the literature (Tables1 and2)[18,21–24,29–33,44,45,48,51–56] All three of our patients display a remarkably short stature. Pt. 2 and 3 are more severely affected, in part because of their kyphoscoliosis, with a stature progressing from moderately short (or normal for Pt. 3) at birth to well below the 3rd percentile during childhood. Nearly all patients reported in the literature (65/66 evaluated cases) share this feature, confirming short stature as a main aspect of spEDS. Joint hypermobility and soft, doughy and hyperextensible skin are some of the most striking characteristics in our patients. These qualities are particularly remarkable in the hands and, to a lesser extent, in the feet, and helped us restrict the diagnosis to an EDS-related condition. Since these features are shared by more than 90% of cases reported in the literature (63/66 and 62/68, respectively), in our opinion their combination should be considered a major diagnostic criterion for spEDS. Craniofacial features for both B4GALT7- and B3GALT6-related conditions are quite variable among our patients and the reported individuals for whom either pictures or descriptive data could be evaluated, and do not seem suggestive of a facial gestalt. There are, however, some recurring features that may sometimes help to set the two conditions apart: i. proptosis is more frequent in spEDS-B4GALT7 (32/33 vs 20/32); ii. midface retrusion has been frequently noted in both conditions, but in spEDS-B3GALT6 it is often associated with a prominent forehead (31/38), sometimes emphasized by sparse hair (12/26) as in the case of our Pt. 2 and 3, while it would be more appropriate to state that spEDS-B4GALT7 patients have an entirely flat face, usually short or round (31/33) and with a narrow mouth (31/33). The progeroid aspect of spEDS-B4GALT7 described in Kresse’s child [24] and previously pointed out by Hernandez [21–23] was absent in all other patients, suggesting that it is not strongly associated with mutations in B4GALT7. As current terminology indicates, a progeroid facial appearance is the subjective interpretation of a series of facial features which should rather be evaluated individually. Some features such as thin skin or excessive wrinkles may occasionally appear in the description of the cases we reviewed, but never in a combination suggestive of premature aging, and none of these were observed in our patient. Therefore, as already suggested by other authors, Genes 2019, 10, 799 16 of 21 we endorse the necessity to remove the term “progeroid type” from this syndrome, because it could be misleading. The radiological findings are undoubtedly the most significant evidence in distinguishing B4GALT7-related and B3GALT6-related spEDS: the latter includes more numerous and pronounced elements of skeletal dysplasia, and for this reason has often been classified as such in the past. Radioulnar synostosis has been observed recurrently and almost exclusively in spEDS-B4GALT7 (19/32 cases, including our patient, vs 3 documented cases of spEDS-B3GALT6 [53,56]. On the other hand, most of the individuals affected by B3GALT6-related spEDS, including our patients, display severe kyphoscoliosis, usually congenital or early onset and progressive (36/39), and several of the skeletal changes associated with SEMDJL1, such as platyspondyly, short iliac bones, elbow dislocation with misalignment of the long bones (33/36). Osteopenia has been noted in both conditions. It has been reported more frequently in B3GALT6-related cases (22/26), usually in conjunction with fractures and luxations, as in the case of our patients. In B4GALT7-related cases there are significantly fewer records of this feature, but in most reports its absence or presence cannot be verified. Therefore, the actual prevalence of osteopenia is unknown and should probably deserve more consideration when referring spEDS suspects for a radiological exam. A consequence of skeletal involvement (bowing of limbs) is considered one of the main criteria for suspecting spEDS, and it is possibly more common in the spEDS-B4GALT7 series (30/32 vs 16/20); in fact, our Pt. 2 and 3 do not have this phenotype. Joint contractures of the upper limbs have been noted for both conditions, though they are usually reported at the elbow in spEDS-B4GALT7 and at the hands in spEDS-B3GALT6. In our experience, it was actually our B3GALT6-mutated patients who displayed limited elbow extension, but this is a non-specific clinical sign that is often found in skeletal dysplasia. Other clinical features appear to be peculiar of B3GALT6-related spEDS. The most notable are talipes equinovarus (23/37 patients, including ours), possibly with hypoplastic nails (cfr EDS classification of 2017 [46] and our patients 2 and 3), and abnormalities of the dentition (13/17 patients, including ours), sometimes with yellow-brown discoloration of the teeth (also observed in our Pt. 2). Motor delay has been noted in the majority of reports in which developmental milestones were evaluated (27/36), including our three patients. It is most likely related to the recurrent skeletal anomalies (bowing of limbs, scoliosis, hip dysplasia) as well as to the frequent reports of hypotonia, also observed in all of our patients (36/48 evaluated cases). In order to overcome the difficulties in sitting and walking, these children often need assistance in the form of rehabilitation with appropriate physiotherapeutic interventions. Depending on the severity of the musculoskeletal features, some individuals may be able to walk without help at a later age than their peers, while others can only manage walking with help. Developmental delay appears to be present in only about half of the documented spEDS cases (26/49). In many instances, psychomotor development is only mildly delayed, possibly because of the confounding effect of minor behavioral abnormalities. Of our three patients, Pt. 1 IQ has been demonstrated to be average, while Pt. 2 and 3 require a support teacher during school only because of a diminished ability to concentrate, and actually have good comprehensive and expressive skills. In summary, among all of these clinical features, the most relevant in order to suspect spEDS, because of their rarity in other syndromic conditions, are: i. the extreme distal joint hypermobility and soft, hyperextensible skin, particularly of the hands; ii. the radiological signs, which are the main indicator for discriminating spEDS-B4GALT7, associated with radioulnar synostosis, and spEDS-B3GALT6, characterized by kyphoscoliosis (congenital or early onset and progressive) and by the skeletal signs of SEMDJL1 (platyspondyly, short iliac bones, elbow dislocation).

Current EDS classification includes a third type of spEDS, caused by biallelic defects in SLC39A13 (OMIM *608735), which encodes a zinc transporter involved in connective tissue development [57]. (Radiological findings suggest a minor skeletal involvement compared to other spEDS, but overall,) Genes 2019, 10, 799 17 of 21 the small number of reported families is inadequate to discuss a differential diagnosis with good confidence [58,59]. The differential diagnosis of spEDS-B3GALT6 may be placed with kyphoscoliotic EDS (kEDS), primarily owing to early onset kyphoscoliosis, but the two conditions are very different from the clinical point of view, because the latter is not characterized by significantly short stature, and the joint hypermobility with dislocations/subluxations involves mainly shoulders, hips and knees rather than hands and feet. The differential diagnosis may be placed also with musculocontractural EDS (mcEDS), but the latter is characterized by congenital multiple contractures, characteristically adduction-flexion contractures, and typical craniofacial features, which are distinct from spEDS. The main differential diagnosis of spEDS-B4GALT7 can be given with arthrochalasia EDS (aEDS), which is characterized by congenital bilateral hip dislocation and laxity of small joints, but generally aEDS also presents subluxation of the knees and dorso-lumbar kyphosis. A clinician should consider the differential diagnosis between spEDS-B4GAL7 and the B3GAT3-related linkeropathy, mainly because of prominent eyes, short stature with joint laxity and radioulnar synostosis. However, it should be noted that patients with B3GAT3 variants often have cardiovascular abnormalities, and that the rarity of the cases described in the literature does not allow definite conclusions [14,15]. Additional clinical descriptions of these disorders are required in order to characterize the linkeropathies both individually and as a disease group. The radiological feature of spEDS may further warrant consideration for differential diagnosis with several other skeletal dysplasias. The main discriminating features of B4GALT7- and B3GALT6-related conditions, i.e. respectively radioulnar synostosis and scoliosis with early onset and rapid evolution, can be found in various skeletal dysplasias, but are associated with such a remarkable degree of joint hypermobility and soft, hyperextensible skin almost exclusively in spEDS.

5. Conclusions The most striking aspect of spEDS is the combination of clinical signs affecting both the connective tissue and the skeletal system. The criteria identified in the International Classification of EDS by Malfait et al. (2017) clearly show that there is significant overlap between the clinical features of spEDS-B4GALT7 and spEDS-B3GALT6. This is confirmed and expanded upon by the clinical reports and literature review tables (Tables1 and2) presented here. Overall, although skin and joints are similarly affected in both conditions, B3GALT6 mutations lead to a more extensive and severe involvement of the skeletal system, with features often found in SEMDJL1 such as kyphoscoliosis, platyspondyly, short iliac bones and elbow disclocation. Careful observation of the hands, with their very soft and hyperextensible skin and extreme distal joint laxity, in combination with the specific radiological signs, can immediately evoke the suspicion of either spEDS-B4GALT7 (radioulnar synostosis) or spEDS-B3GALT6 (severe progressive kyphoscoliosis and other SEMDJL1-like skeletal features). The extreme hypermobility of distal joints and the soft, doughy skin on the hands and feet are rarely seen in other EDS types (except, to some degree, in kEDS and aEDS) and are a valuable clue for the diagnosis, which should be supported by skeletal radiographs and by molecular analysis. Once spEDS is suspected, direct Sanger sequencing is still a viable and cost-effective option for molecular analysis, since B4GALT7 and B3GALT6 have short coding regions and only a few exons (6 and 1, respectively). This should be considered especially if the radiological signs restrict the hypothesis to either gene. However, particularly when attempting a molecular analysis early on, when some of the distinctive clinical signs have not yet evolved, an NGS panel specific for EDS would be advisable, since it can help confirm the differential diagnosis. Being able to distinguish between spEDS-B3GALT6 and spEDS-B4GALT7 is important for clinicians, because some patients in the B3GALT6-related group may develop life-threatening complications such as aortic dilatation, aneurysms and cervical spine instability. Genes 2019, 10, 799 18 of 21

In conclusion, accurate diagnosis will help in excluding other causes of peripheral hypotonia, such as neuromuscular disorders, and allow for appropriate physiotherapeutic interventions.

Author Contributions: L.G. and A.S.-F conceived the study. L.G., I.M., I.I., M.P. M.D.R., C.M. and S.G. performed the clinical diagnosis of the patient, genetic counselling and follow-up; M.V. and A.R. performed biochemical studies in skin fibroblasts; S.S. and S.F. investigated bone health parameters; B.C-X. and S.U. carried out the molecular analyses and interpreted the results; I.I. and S.G.C. researched the literature; L.G, I.I., and S.G.C. prepared the manuscript; A.S.F. edited and coordinated the manuscript. All authors discussed, read, and approved the manuscript. Funding: This research received no external funding. Acknowledgments: The authors also wish to thank the patients’ family members for their cooperation in providing the medical data and photographs necessary for this publication, as well as the photographers Marco Bonazzi and Luca Valcavi. The authors are grateful for the contribution made by the Fondazione Cassa di Risparmio Manodori of Reggio Emilia. Conflicts of Interest: All authors declare that there are no conflicts of interest concerning this work.

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